LOCUS       sequence-350092-        6502 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding MRAP and EmGFP-Bsd.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6502)
  AUTHORS   Moyer AJ, Fernandez FX, Li Y, Klinedinst DK, Florea LD, Kazuki Y, 
            Oshimura M, Reeves RH
  TITLE     Overexpression screen of chromosome 21 genes reveals modulators of 
            Sonic hedgehog signaling relevant to Down syndrome.
  JOURNAL   Dis Model Mech. 2023 Apr 1;16(4):dmm049712. doi: 10.1242/dmm.049712.
            Epub 2023 Apr 13.
  PUBMED    36995257
REFERENCE   2  (bases 1 to 6502)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Dis Model 
            Mech."; date: "2023-04-1"; volume: "16(4):dmm049712. doi"; pages: " 
            10.1242/dmm.049712. Epub 2023 Apr 13"
COMMENT     Sequence Label: MRAP_pcDNA6.2/EmGFP-Bsd
FEATURES             Location/Qualifiers
     source          1..6502
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    911..935
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1458..1482)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1508..1549
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(1589..1608)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1633..1681
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1883..2311
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1970..1989)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2180..2201
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2326..2825
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2361..2384)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2758..2777
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        2839..2886
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(2905..2907,2908..2910,2911..3621)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2950..2971)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3211..3230)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3558..3579
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3622..4017
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    4175..4296
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4212..4231)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4266..4285
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4345..4361)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4345..4361)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4358..4380)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4369..4385
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4393..4399,4400..4417,4418..4423))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4438..4459
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4576..4593)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4747..5335)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4827..4846)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5506..6297,6298..6366))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPKLNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6129..6148
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6367..6471)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagt
      901 taagctatca acaagtttgt acaaaaaagc aggcaccatg gccaacggga ccaacgcctc
      961 tgccccatac tacagctatg aatactacct ggactatctg gacctcattc ccgtggacga
     1021 gaagaagctg aaagcccaca aacattccat cgtgatcgca ttctgggtta gcctggctgc
     1081 cttcgtggtg ctgctcttcc tcatcttgct ctacatgtcc tggtccgcct ccccgcagat
     1141 gaggaacagc cccaagcacc accaaacatg cccctggagt cacggcctca acctccacct
     1201 ctgcatccag aagtgcctgc cgtgccacag ggaacccctg gcaacctcac aggctcaggc
     1261 gagctcagtg gagccaggga gcagaactgg ccctgaccag ccgctacgac aggagagctc
     1321 ctccacgttg cccctcgggg gtttccagac ccaccccact ctcctctggg aactgaccct
     1381 caatgggggt cccctcgtca ggagcaagcc cagcgagcct ccccctggag acaggacctc
     1441 tcaattgcag agctagaacc cagctttctt gtacaaagtg gttgatctag agggcccgcg
     1501 gttcgaaggt aagcctatcc ctaaccctct cctcggtctc gattctacgc gtaccggtta
     1561 gtaatgagtt taaacggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa
     1621 cccgcgctat gacggcaata aaaagacaga ataaaacgca cgggtgttgg gtcgtttgtt
     1681 cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat
     1741 tggggccaat acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa
     1801 ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg ccatagcaga tctgcgcagc
     1861 tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     1921 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     1981 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     2041 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     2101 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     2161 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     2221 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     2281 gagctgattt aacaaaaatt taacgcgaat tatcctcgag ctaccgggta ggggaggcgc
     2341 ttttcccaag gcagtctgga gcatgcgctt tagcagcccc gctgggcact tggcgctaca
     2401 caagtggcct ctggcctcgc acacattcca catccaccgg taggcgccaa ccggctccgt
     2461 tctttggtgg ccccttcgcg ccaccttcta ctcctcccct agtcaggaag ttcccccccg
     2521 ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc actagtctcg
     2581 tgcagatgga cagcaccgct gagcaatgga agcgggtagg cctttggggc agcggccaat
     2641 agcagctttg ctccttcgct ttctgggctc agaggctggg aaggggtggg tccgggggcg
     2701 ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga ggcccggcat
     2761 tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca tctccgggcc
     2821 tttcgactct agacacgtgt tgacaattaa tcatcggcat agtatatcgg catagtataa
     2881 tacgacaagg tgaggaacta aaccatggtg agcaagggcg aggagctgtt caccggggtg
     2941 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     3001 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     3061 aagctgcccg tgccctggcc caccctcgtg accaccttca cctacggcgt gcagtgcttc
     3121 gcccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     3181 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     3241 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     3301 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caaggtctat
     3361 atcaccgccg acaagcagaa gaacggcatc aaggtgaact tcaagacccg ccacaacatc
     3421 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     3481 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
     3541 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     3601 ggcatggacg agctgtacaa ggccaagcct ttgtctcaag aagaatccac cctcattgaa
     3661 agagcaacgg ctacaatcaa cagcatcccc atctctgaag actacagcgt cgccagcgca
     3721 gctctctcta gcgacggccg catcttcact ggtgtcaatg tatatcattt tactggggga
     3781 ccttgtgcag aactcgtggt gctgggcact gctgctgctg cggcagctgg caacctgact
     3841 tgtatcgtcg cgatcggaaa tgagaacagg ggcatcttga gcccctgcgg acggtgccga
     3901 caggtgcttc tcgatctgca tcctgggatc aaagccatag tgaaggacag tgatggacag
     3961 ccgacggcag ttgggattcg tgaattgctg ccctctggtt atgtgtggga gggctaagca
     4021 cttcgtggcc gaggagcagg actgacacgt gctacgagat ttcgattcca ccgccgcctt
     4081 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     4141 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
     4201 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     4261 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
     4321 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     4381 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     4441 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     4501 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     4561 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     4621 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     4681 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     4741 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     4801 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     4861 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     4921 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     4981 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     5041 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     5101 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     5161 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     5221 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     5281 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     5341 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     5401 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     5461 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     5521 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     5581 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     5641 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     5701 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     5761 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     5821 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct tcggttccca
     5881 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     5941 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     6001 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     6061 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     6121 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     6181 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     6241 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     6301 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     6361 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     6421 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     6481 ccgaaaagtg ccacctgacg tc
//
